Transplantation of autologous corneal epithelial stem/progenitor cells (CESCs) expanded in tissue culture has successfully restored vision and revolutionized the treatment for limbal stem cell deficiency (LSCD) which is a major cause, either primary or secondary, of significant visual loss and blindness in many common corneal disorders. A higher expansion efficiency of the stem/progenitor cell population in culture corresponds to a better long-term graft survival. The most efficient expansion method requires mouse 3T3 feeder cells which are grown using calf serum and are the source of mouse RNA that contaminates the expanded cells. This cross-contamination by animal products poses considerable potential health hazards and therefore makes this culture method unlikely to be approved by the US Food and Drug Administration to be used in humans. New cell engineering methods that achieve the same or better efficiency of expanding CESCs under xenobiotic-free conditions are needed to achieve acceptable clinical outcomes. The long-term goal of my laboratory is to elucidate the regulatory factors that govern CESC self-renewal and differentiation, and to develop patient- specific stem cell-based therapies for LSCD. The objective of this particular application is to identify optimal cell engineering systems that can specifically and efficiently expand the stem/progenitor cell population of human corneal epithelial cells for transplantation. The central hypothesis is that appropriate human feeder cells can replace mouse 3T3 cells to provide a proper microenvironment to support the growth of CESCs, and upon receiving additional appropriate external signals the expansion of CESCs could be further optimized in culture. The hypothesis has been formulated based on the data produced in my laboratory. To achieve the objective of this translational research application, two specific aims are proposed: 1) Establish a xenobiotic-free culturing system using a human feeder layer that can efficiently expand CESCs; and 2) Identify an optimal expansion condition of CESCs by modulating the Wnt and/or Notch signaling pathway using small molecules or bioengineered human feeder cells.) Under the first aim, five human feeder candidates will be tested for their ability to grow CESCs. The functional aspect of these bioengineered CESCs will be tested in a well-established mouse model of LSCD. Under the second aim, proliferation of CESCs will be further optimized using human feeder cells that are engineered to over express limbal-specific Wnt molecules and Notch ligands, and small molecules of Wnt activators and Notch inhibitors. The approach is innovative, because it utilizes a novel method to bioengineer CESCs without permanent genetic alternation of the target cells. This approach has three major advantages: reversible, specific and translational. This will eliminate any potential permanent side effects or toxicity. The proposed translational research is significant, because the results from any of the two aims can be readily adapted for clinical development.